CN114717224B - Method for enriching and identifying glycosylated ribonucleic acid glycoRNA based on solid phase - Google Patents
Method for enriching and identifying glycosylated ribonucleic acid glycoRNA based on solid phase Download PDFInfo
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Abstract
The invention discloses a method for enriching and identifying glycosylated ribonucleic acid (RNA) based on a solid phase, which comprises the steps of firstly, extracting total RNA from a biological/clinical sample, and oxidizing glycan carried by the RNA; then, covalently binding oxidized glycoRNA to the solid phase; then, eluting the non-covalently adsorbed RNA; finally, different glycosidases are used to gradually obtain different types of glycornas. The invention can identify the types and the quantity of the N-glycoRNA and the O-glycoRNA, and lays a foundation for the mechanism elucidation, development and application of disease diagnosis markers.
Description
Technical Field
The invention belongs to the technical field of biological molecular analysis reagents, and particularly relates to a method for enriching and recognizing glycosylated ribonucleic acid glycoRNA in a solid phase.
Background
Glycosylation modification can regulate a plurality of important physiological functions in cells, such as correct folding, transportation, degradation and the like of proteins, which are not involved in glycosylation; glycoproteins and glycolipids on the cell membrane also play an indispensable role in the communication process between cells. Thus, the conventional wisdom holds that proteins and lipids are the major target macromolecules for glycosylation modification, and that glycoproteins and glycolipids on the cell membrane play an important role in the transfer of information between cells, but this wisdom has been altered at present. Carolyn R.Bertozzi and Ryan A.Flynn scientific research team from Stanford university chemical series in 2021 first proposed that ribonucleic acid (RNA) is the third largest target macromolecule for glycosylation (Cell 2021,184 (12)). Through studies on H9 and HeLa cells, RNA was found to be glycosylated with N-glycans, called N-glycoRNA. And experiments confirm that they belong to the class of small RNAs including YRNA, micronuclear RNAs (snrnas), ribosomal RNAs (rrnas), nucleolar micrornas (snoRNAs) and transfer RNAs (tRNAs). Currently, these glycornas are demonstrated to be mainly enriched outside the cell membrane of living cells, and thus presumably have similar functions to glycoproteins and lipids on the cell membrane. Indeed, RNA has been found to have a variety of post-translational modifications, such as acetyl RNA. However, glycoRNA glycosylation modifications have been shown to be on the surface of cell membranes, possibly with important biological functions, and have received attention from the global scientific community since discovery. Further results of the research group indicate that glycornas on these cell membranes can be recognized not only by RNA antibodies, but also as direct ligands for immunoglobulin-like lectin (Siglec) receptors and play important physiological functions in immune regulation. The scientific research team mainly adopts a DBCO-biotin copper-free click chemistry method to identify the glycoRNA, and uses a Northern blot technology combined with poly-A magnetic bead enrichment to infer the length of the glycoRNA (less than 200 nt). And separating the glycoRNA by combining a super-high speed centrifugation method and carrying out subsequent sequencing. However, the azide (Ac 4 ManNAz) used for labeling sugar has the advantages of high toxicity, easy explosion, low specificity, and time-consuming and difficult extraction by sucrose gradient centrifugation. The method for identifying and identifying the glycoRNA in the team is complex, has high requirements on instruments and equipment, has certain requirements on operators, and brings difficulty to subsequent research, preparation and production.
Thus, there is a need to develop a rapid, accurate, safe and low cost method for enriching and identifying glycornas.
Disclosure of Invention
The invention aims to provide a method for enriching and identifying glycosylated ribonucleic acid (RNA) based on a solid phase.
The technical scheme of the invention is as follows:
a method for enriching glycosylated ribonucleic acid glycoRNA based on a solid phase, comprising the steps of:
(1) Extracting total RNA:
(1) adding Trizol reagent into the sample, adding chloroform, shaking, mixing, and standing on ice;
(2) centrifuging, absorbing supernatant, adding equal volume of isopropanol, shaking up and down, mixing, and standing at room temperature;
(3) centrifuging, removing supernatant, and retaining precipitate;
(4) washing the precipitate with ethanol multiple times, centrifuging at room temperature, and removing liquid in the tube;
(5) drying at room temperature, dissolving the precipitate with DEPC water to obtain total RNA solution;
(2) Preparation of glycoRNA:
(1) adding an oxidant into the total RNA solution and incubating to obtain oxidized RNA;
(2) measuring resin according to the quality of total RNA obtained in the step (1), transferring the resin into a centrifuge tube filter tube without RNase, washing the resin for a plurality of times by using DEPC water, adding the oxidized RNA, and incubating;
(3) removing liquid in the centrifugal filter tube, and washing the centrifugal filter tube with DEPC water for multiple times to ensure that the eluent does not contain RNA;
(4) sealing the bottom of the centrifugal filter tube, adding N-endonuclease and buffer solution into the centrifugal filter tube, incubating, collecting eluent N-glycoRNA, and preserving at-80 ℃;
(5) eluting the resin for multiple times by using DEPC water to ensure that the eluent does not contain RNA;
(6) adding O-glycosidase and buffer solution into the centrifugal filter tube, incubating, collecting eluent which is O-glycoRNA, and preserving at-80 ℃.
Further, after step (5) of step (1), the method further comprises the steps of:
(6) measuring the concentration and optical density value of the total RNA solution by using a Nanodrop instrument;
(7) preparing agarose solution, putting the agarose solution into a microwave oven for treatment, cooling to obtain agarose gel solution, uniformly mixing GelGreen dye into the agarose gel solution, pouring the uniformly mixed agarose gel solution into a slab rubber mold, cooling, putting into an electrophoresis buffer solution, loading samples, and extracting total RNA;
(8) the integrity of the total RNA was checked with a bioanalyzer.
Further, in the step (6), the concentration of the total RNA solution and the A260/A280 of the optical density value are 1.8-2.0, and the A260/A230 is 2.1-2.3.
Further, in the step (7), the agarose solution is composed of agarose and 1 Xethylenediamine tetraacetic acid buffer, the ratio of the agarose to the 1 Xethylenediamine tetraacetic acid buffer is 1g to 100mL, the total RNA mass in the loaded sample is 200-1000ng, the extracted total RNA satisfies that the brightness of 28S is twice the brightness of 18S, and the band is not diffused.
Further, in step (8), RIN > 9.
Further, in step (2) of step (2), the ratio of the mass of the total RNA to the volume of the resin is 1. Mu.g: 4. Mu.L.
Further, in step (4) of step (2), the ratio of the mass of the total RNA to the N-endonuclease is 5. Mu.g: 1 mul.
Further, in step (6) of step (2), the ratio of the mass of the total RNA to the O glycosidase is 5. Mu.g: 1 mul.
The other technical scheme of the invention is as follows:
a method for solid phase-based recognition of glycosylated ribonucleic acid glycoRNA comprising the steps of:
(1) Obtaining N-glycoRNA and O-glycoRNA by a method based on solid phase enrichment of glycosylated ribonucleic acid glycoRNA;
(2) Quality detection and sequencing of Small RNAs:
(1) respectively detecting the length distribution of RNA in the N-glycoRNA and O-glycoRNA solution by adopting a biological analyzer, and detecting quality qualified when a peak exists in 40 nt;
(2) after quality inspection is qualified, constructing a sample library, and when the concentration of qPCR is more than 3nM, constructing the library successfully, and then performing small RNA sequencing.
Further, after the quality detection and sequencing of the Small RNA in step (2), the method further comprises:
(3) Bioinformatics analysis:
(1) performing basic data processing on the sequencing result of the smalls RNA sequencing: image recognition, base recognition, filtering the linker sequence, removing low quality sequences;
(2) advanced data processing: statistical small RNA length distribution, public sequence and specific sequence analysis comparing normal and disease group samples, identification of known microRNAs, prediction of new microRNAs, small RNA annotation, downstream analysis of two sample differential target genes and visualization.
The invention provides a method for enriching and identifying glycosylated ribonucleic acid (RNA) based on a solid phase, which can simply and rapidly prepare the RNA, and can be used for qualitative and quantitative analysis of the RNA in normal cells and abnormal cells and search of glycosylation sites; complicated experimental steps, toxic reagents and expensive instruments can be avoided; the processing efficiency of the sample is improved, and the method can be used for high-flux sample processing to find differential glycoRNA; the distinction of N-glycoRNA and O-glycoRNA lays a foundation for the research of subsequent mechanisms, the discovery of biomarkers and the like.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the description of the embodiments will be briefly described below, it being obvious that the drawings in the following description are only some embodiments of the present invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art. Wherein,
FIG. 1 is a schematic of a workflow for the enrichment of glycosylated RNA by solid phase in the preparation of glycoRNA according to the present invention, wherein square shading represents N-acetylglucosamine, circular shading represents glucose, circular shading represents galactose, square shading represents sialic acid, and triangular shading represents fucose;
FIG. 2 is a schematic representation of total RNA extraction in the present invention;
FIG. 3 is a schematic representation of glycoRNA oxidation in accordance with the present invention;
FIG. 4 is a schematic representation of the oxidized N-glycoRNA of the present invention bound to a solid phase bearing an amino group and resulting in N-glycoRNA;
FIG. 5 is a schematic representation of the oxidized O-glycoRNA of the present invention bound to a solid phase bearing an amino group and resulting in O-glycoRNA;
FIG. 6 is a graph showing agarose gel electrophoresis results of M1 and M2 samples according to the present invention;
FIG. 7 is a graph showing the quality inspection result of an M1 sample in Agilent 2200 in the invention;
FIG. 8 is a graph showing the quality inspection results of an M1 sample in Agilent 2100 according to the present invention;
FIG. 9 is a library fragment profile of sample M1 in Agilent 2100 according to the present invention;
FIG. 10 is a graph showing the distribution of the lengths and types of small RNA in M1 and M2 samples according to the present invention.
Detailed Description
The invention develops a method for enriching and identifying glycosylated ribonucleic acid (RNA) based on a solid phase, referring to FIG. 1, FIG. 1 is a schematic diagram of a workflow for preparing glycosylated RNA by enriching glycosylated RNA in the solid phase. As shown in figure 1, after total RNA is extracted, the total RNA is oxidized to obtain glycoRNA with aldehyde groups, the glycoRNA is covalently bound with hydrazide or amine groups to be enriched on resin, and finally, N-glycoRNA or glycoRNA containing GalNAc and GalGalNAc is obtained by enzymatic hydrolysis. The method specifically comprises the following steps: extracting total RNA and checking quality; enrichment and preparation of glycoRNA; small RNA quality detection and sequencing; glycoRNA bioinformatics analysis.
Through the method, glycan in the glycoRNA is oxidized, the glycoRNA is coupled to solid-phase resin (bead) with amino or hydrazide, N-glycosidase and O-glycosidase are used for respectively performing enzyme digestion, respective eluents are collected, the types and the amounts of the N-glycosylation and the O-glycosylation glycoRNA are respectively identified, and a foundation is laid for the development of follow-up disease biomarkers and the elucidation of mechanisms.
In order to make the above objects, features and advantages of the present invention more comprehensible, the following technical solutions of the present invention are further described with reference to the accompanying drawings and examples. The invention is not limited to the embodiments listed but includes any other known modification within the scope of the claims that follow.
First, reference herein to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic can be included in at least one implementation of the invention. The appearances of the phrase "in one embodiment" in various places in the specification are not necessarily all referring to the same embodiment, nor are separate or alternative embodiments mutually exclusive of other embodiments.
In the following detailed description of the embodiments of the present invention, the schematic drawings are not to be taken in a local scale for the convenience of description, and are merely examples, which should not limit the scope of the present invention. In addition, the three-dimensional space of length, width and depth should be included in actual fabrication.
Finally, the water involved in the present invention was DEPC (deionized and diethylpyrocarbonate) water, and the instruments and tubes, pipette guns, etc. used were treated with RNase removal reagent. M refers to concentration mol/L, and the used reagent is prepared as follows:
1M NH 4 HCO 3 ＝79.06mg NH 4 HCO 3 +1mL DEPC water
25mM NH 4 HCO 3 ＝40μL 1M NH 4 HCO 3 +1560. Mu.L DEPC Water
20% dmso = 120 μl+480 μl DEPC water
70% ethanol=7ml ethanol+3 mL DEPC water
Example 1
A method for enriching and recognizing glycosylated ribonucleic acid glycoRNA based on a solid phase, comprising the steps of:
(1) Extraction and quality control of total RNA in cells
The conventional Trizol method is used herein to extract total RNA from human or animal derived tissues or cells (collectively referred to as samples). Referring to fig. 2, fig. 2 is a schematic diagram showing the extraction of total RNA in the present invention. As shown in FIG. 2, RNA was extracted by Trizol method after dissolving the sample in chloroform, precipitating total RNA by adding isopropanol to the aqueous phase (pH < 7) after delamination, washing the total RNA with 70-75% ethanol, precipitating at least 2 times, and finally dissolving the total RNA precipitate with DEPC water to obtain total RNA solution. The steps are as follows:
(1) trizol reagent (Beyozol, shanghai) (5X 10) was added to the sample 6 Cells/1 mL Trizol;50-100mg tissue homogenate/1 mL Trizol) and transferred toAdding 200 mu L of chloroform into a 1.5mL centrifuge tube, shaking and mixing for 30s, and standing on ice for 3min;
(2) a centrifuge (cooling the system to the required temperature in advance) at 4 ℃ for 12000g, centrifuging for 15min, sucking 400 mu L of supernatant, adding equal volume (400 mu L) of isopropanol, shaking up and down, mixing, and standing for 15min at room temperature;
(3) centrifuging at 4deg.C under a centrifugal force of 12000g for 15min, and removing supernatant as much as possible (double gun head method);
(4) adding 700 mu L of 70-75% ethanol, washing the total RNA precipitate at least 2 times, centrifuging at room temperature at 8000g for 5min, and removing liquid in the centrifuge tube;
(5) after drying at room temperature for 5min, the precipitate was dissolved with 20 μl DPEC water to give a total RNA solution;
(6) measuring total RNA concentration and Optical Density (OD) values with a Nanodrop instrument, wherein A260/A280 is 1.8-2.0, and A260/A230 is 2.1-2.3;
(7) agarose gel electrophoresis characterization: 50mL of a 1% (w/v) agarose solution (0.5 g agarose and 50mL 1 XTAE) was prepared, treated in a microwave oven for 1.5-2min (30 s initially, mixed every 10 s), cooled to 60-70℃and 5. Mu.l of GelGreen dye (Beyotidme, shanghai) was added and mixed homogeneously in a 1% agarose gel solution. Pouring the well mixed agarose gel solution into a gel plate mould (avoiding air bubbles), cooling for about 30min, and placing into an electrophoresis buffer solution and loading (the total RNA mass is 200-1000 ng). Setting the voltage to 110V and running for 20-30min. The total RNA extracted was required to meet 28S brightness of twice 18S with no band spread;
(9) total RNA integrity (RIN > 9) was checked using an Agilent 2200 bioanalyzer.
(2) Preparation of GlycoRNA
Referring to FIG. 3, FIG. 3 is a schematic diagram of glycoRNA oxidation according to the present invention. Gal and GalNAc are oxidized to form aldehyde groups. As shown in the figure 3 of the drawings, the method comprises the following steps:
(1) sequentially adding a certain amount of oxidant to the total RNA solution and incubating for 2-3h (ph=7.0-7.2) to obtain oxidized RNA;
(2) measuring a certain amount of resin according to the mass of total RNA (the mass of total RNA: the volume of resin is approximately 1 mug: 4 mug), transferring the resin into a centrifugal filter tube without RNase, washing the resin twice by using DEPC water, adding oxidized RNA, and incubating for 1-2h at 25 ℃;
(3) removing liquid in the centrifugal filter tube, and cleaning with 500 mu L DEPC water for three times to ensure that the eluent does not contain RNA;
(4) referring to FIG. 4, FIG. 4 is a schematic diagram showing the binding of oxidized N-glycoRNA to a solid phase with amino groups and obtaining N-glycoRNA according to the present invention. As shown in fig. 4, the bottom of the centrifugal filtration tube was sealed, and a certain amount of N-endonuclease (mass of total RNA: N-endonuclease ≡ 5. Mu.g: 1. Mu.L) and corresponding buffer (pH=7.0-7.2), such as 25mM ammonium bicarbonate solution (NH 4 HCO 3 ) Or 25mM Tris-HCl, incubating for 1-2h at 25 ℃, collecting eluent N-glycoRNA, and preserving at-80 ℃;
(5) eluting the resin with 500 μl DEPC water for three times to ensure that the eluate contains no RNA;
(6) referring to FIG. 5, FIG. 5 shows the binding of oxidized O-glycoRNA to a solid phase with amino groups and obtaining O-glycoRNA according to the present invention. As shown in FIG. 5, an amount of O-glucosidase (mass of total RNA: O-glucosidase. Apprxeq.5. Mu.g: 1. Mu.L) and a corresponding buffer (pH=7.0-7.2) such as 25mM ammonium bicarbonate solution (NH) 4 HCO 3 ) Or 25mM Tris-HCl,25 ℃, incubating for 1-2h, and collecting the eluent as O-glycoRNA. Stored at-80 ℃.
The following steps can be used to detect whether glycoRNA preparation was successful
(3) Quality detection and sequencing of Small RNA
(1) Respectively detecting the length distribution of RNA in the N-glycoRNA and O-glycoRNA solution obtained in the steps by adopting an Agilent 2100 bioanalyzer, wherein the requirement of 40nt peak-presence is met;
(2) after quality inspection is qualified, constructing a sample library and requiring the concentration of qPCR to be more than 3nM;
(3) when the library was constructed successfully, small RNA sequencing was performed again.
(4) Bioinformatics analysis
(1) Basic data processing of sequencing results: image recognition, base recognition, filtering the linker sequence, removing low quality sequences;
(2) advanced data processing: statistical small RNA length distribution, public sequence and specific sequence analysis comparing normal and disease group samples, identification of known microRNAs, prediction of new microRNAs, small RNA annotation, downstream analysis of two sample differential target genes and visualization.
Example 2
Referring to FIG. 1, FIG. 1 is a schematic diagram showing the workflow of the present invention for the preparation of glycosylated RNA by solid phase enrichment. As shown in FIG. 1, a solid phase glycosylated RNA enrichment and N-endonuclease based analytical method (SPRNA-N) and O-glycosidase analytical method (SPRNA-O) comprising the steps of:
(1) Extraction and quality inspection of Total RNA:
the method comprises the following steps: tissue RNA extraction
A biopsy sample of a clinical patient or animal was collected, 250mg of tissue was taken, rapidly ground in liquid nitrogen, transferred to an EP tube of RNase-free, 1mL of Trizol reagent (Beyozol, shanghai) was added, 200. Mu.L of chloroform was further added, and the mixture was shaken and mixed for 30s, and left on ice for 3min. Put in a centrifuge at 4 ℃,12000g, centrifuge for 15min, suck up 400 μl of supernatant, add equal volume (400 μl) of isopropanol, mix upside down, stand at room temperature for 15min. And putting the mixture into a centrifuge with the temperature of 4 ℃ for 12000g, centrifuging for 15min, and removing the supernatant. After adding 700. Mu.L of 70% ethanol to the centrifuge tube, rinsing the RNA pellet at least 2 times, and centrifuging at 8000g for 5min at room temperature, removing the liquid from the centrifuge tube, drying for 5min at room temperature, and dissolving the pellet with 20. Mu.L of DPEC water.
The second method is as follows: cellular RNA extraction
After washing Mia-paca cells 3 times with 1 XPBS solution, 1mL Trizol (about 5X 10) 6 Cells) and transferred to an EP tube, 200 μl of chloroform was added, mixed well with shaking for 30s, and left on ice for 3min. Put in a centrifuge at 4 ℃,12000g, centrifuge for 15min, suck 400 μl of supernatant, add equal volume (400 μl) of isopropanol, mix upside down, stand at room temperature for 15min. And putting the mixture into a centrifuge with the temperature of 4 ℃ for 12000g, centrifuging for 15min, and removing the supernatant. Re-feeding into centrifuge tubeAfter adding 700. Mu.L of 70% ethanol, the RNA pellet was rinsed (n.gtoreq.2) and centrifuged at 8000g for 5min at room temperature, the liquid in the centrifuge tube was removed. The precipitate was dried at room temperature for 5min and dissolved with 20. Mu.L DPEC water.
Total RNA obtained from method one (sample 1) and method two (sample 2) was assayed for RNA purity using Nanodrop assay for RNA concentration and OD, 1% agarose gel electrophoresis was tested for degradation of RNA, and agilent 2200 bioanalytical instrument was used to quality check RNA integrity.
(2) Preparation of N-glycosylated GlycoRNA
(1) Oxidation of glycosylated RNA
Nanodrop measures total RNA concentration, takes 20-40. Mu.g total RNA and adds 100-200. Mu.L of 20% DMSO, 2. Mu.L of horseradish peroxide (HRP), 10-20M galactose oxidase (GAO), 25℃and incubates for 2-3h (pH=7 or so).
(2) Binding to solid phase
80. Mu.L of hydrazide resin (Thermo Fisher Scientific, MA, USA) was measured per 20-40. Mu.g total RNA and transferred to RNase-free centrifuge filter tubes. The hydrogel resin was washed twice with DEPC water and the oxidized RNA from step (1) was added and incubated for 1-2h at 25 ℃.
(3) Preparation of N-glycosylated RNA
The liquid in the centrifugal filter tube in the step (2) is removed, and the centrifugal filter tube is washed with 500 mu L of DEPC water for three times, so that the concentration of RNA in the eluent is ensured to be 0. The bottom of the centrifugal filtration tube was sealed using a rubber cap, and 200. Mu.L of 25mM NH was added to the tube 4 HCO 3 (ph=7), 10-20m PNGase f,25 ℃, incubated for 2h, and the eluate was collected and stored at-80 ℃.
(4) Preparation of O-glycosylated GlycoRNA
The centrifuge filter tube in step 2 was washed three times with 500 μl DEPC water and the RNA concentration in the eluate was guaranteed to be 0. 200. Mu.L of 25mM Tris-HCl (pH=7), 10-20M GalNAcEXO,25℃were added thereto, incubated for 1-2 hours, and the eluate was collected and stored at-80 ℃.
(3) Small RNA sequencing
(1) small RNA quality inspection
Agilent 2100 was used to detect the concentration and length distribution of glycoRNA obtained in steps (2) (3) and (4).
(2) small RNA pooling and sequencing
And (3) after the quality inspection in the step (1) is qualified, performing small RNA library construction, and performing on-machine sequencing after the library construction is successful.
(4) Bioinformatics analysis
Sequencing results, using Cutadapt, fastqc, bowtie2, mirdieep 2, miranda, DESeq, edgeR, GOseq, etc. software and R package, were run on Linux systems for downstream analysis and visualization.
(5) Preliminary results
By the above method, N-glycosylated RNAs (N=2, M1 and M2) in human pancreatic cells (sample 1: mia-paca) were successfully identified, and partial data results are shown below:
1. total RNA quality inspection
1% agarose gel
Referring to FIG. 6, FIG. 6 is a graph showing agarose gel electrophoresis results of M1 and M2 samples according to the present invention, wherein lane 1 is a 1kb DNA maker; lanes 2 and 3 are total RNA extracted from Mia-paca. As shown in fig. 6, the results indicate that 28S is twice as bright as 18S and no significant degradation occurs.
2. Agilent 2200 biological analyser quality control
Referring to fig. 7, fig. 7 is a graph showing the quality inspection result of the M1 sample in agilent 2200 according to the present invention. As shown in FIG. 7, the RINe values of the total RNA of M1 and M2 were 10 and 9.8, respectively.
3. Results of Nanodrop instrument: a260/a280=1.96, a260/a230=2.1
Based on the preliminary results, the quality of two total RNAs (sample 1) extracted from human pancreas cells (Mia-paca) at the time can be judged to be qualified, and the total RNAs can be used for the next experiment.
1. Small RNA quality inspection (Agilent 2100)
Referring to fig. 8, fig. 8 is a graph showing the quality inspection result of the M1 sample in Agilent 2100 according to the present invention. As shown in fig. 8, agilent 2100 results showed that sample M1 was higher in concentration and contained a major peak, allowing library construction.
2. Library construction
Referring to FIG. 9, FIG. 9 is a graph showing the distribution of M1 samples in Agilent 2100 according to the present invention. As shown in FIG. 9, agilent 2100 quality inspection results show that the library fragment is single, the main peak is 147bp and 162bp, and the on-line requirement is met. QPCR results showed that this sample M1 concentration was 18.85nM.
3. Sequencing results
Referring to FIG. 10, FIG. 10 is a graph showing the distribution of the lengths and types of small RNA in M1 and M2 samples according to the present invention. As shown in FIG. 10, the results of the present invention are consistent with those of Carolyn R.Bertozzi and Ryan A.Flynn scientific teams, and rRNA, snRNA, snoRNA and tRNA's were also identified by the present invention. Furthermore, using this method, the micrornas were also identified to have N-glycosylation.
Compared with the prior art, the invention has the beneficial effects that: the invention provides a method for enriching and identifying glycosylated ribonucleic acid (RNA) based on a solid phase, which is characterized in that glycosylated RNA is enriched from different RNA biological samples through the reaction of RNA with amino solid phase and oxidized RNA with aldehyde group, and the types and abundance of the N-glycosylated RNA and the O-glycosylated RNA are respectively identified by utilizing the specificity of endoglycosidase (N sugar and O sugar), thereby laying a foundation for the mechanism elucidation, development and application of disease diagnosis markers.
It should be noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the same, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solution of the present invention may be modified or substituted without departing from the spirit and scope of the technical solution of the present invention, which is intended to be covered in the scope of the claims of the present invention.
Claims (1)
1. A method for enriching glycosylated ribonucleic acid glycoRNA based on a solid phase, comprising the steps of:
(1) Extracting total RNA:
(1) adding Trizol reagent into the sample, adding chloroform, shaking, mixing, and standing on ice;
(2) centrifuging, absorbing supernatant, adding equal volume of isopropanol, shaking up and down, mixing, and standing at room temperature;
(3) centrifuging, removing supernatant, and retaining precipitate;
(4) washing the precipitate with ethanol multiple times, centrifuging at room temperature, and removing liquid in the tube;
(5) drying at room temperature, dissolving the precipitate with DEPC water to obtain total RNA solution;
(6) measuring the concentration and optical density value of the total RNA solution by using a Nanodrop instrument, wherein the A260/A280 of the concentration and optical density value of the total RNA solution is 1.8-2.0, and the A260/A230 is 2.1-2.3;
(7) preparing agarose solution, putting the agarose solution into a microwave oven for treatment, cooling to obtain agarose gel solution, uniformly mixing GelGreen dye into the agarose gel solution, pouring the uniformly mixed agarose gel solution into a slab mold, cooling, putting into an electrophoresis buffer solution and loading the electrophoresis buffer solution into a sample to extract total RNA, wherein the agarose solution consists of agarose and 1 x ethylenediamine tetraacetic acid buffer solution, the ratio of the agarose to the 1 x ethylenediamine tetraacetic acid buffer solution is 1g to 100mL, the mass of total RNA in the loaded sample is 200-1000ng, the brightness of the extracted total RNA meeting 28S is twice that of 18S, and the strip is free from diffusion;
(8) detecting the integrity of the total RNA with a bioanalyzer, wherein RIN > 9;
(2) Preparation of GlycoRNA
(1) Oxidation of glycosylated RNA
Nanodrop detects total RNA concentration, takes 20-40 μg total RNA and adds 100-200 μl 20% DMSO,2 μl horseradish peroxidase, 10-20 μm galactose oxidase, 25 ℃ and incubate for 2-3h, pH=7;
(2) binding to solid phase
Measuring 80 mu L of hydrazide resin per 20-40 mu g total RNA, transferring to an RNase-free centrifugal filter tube, washing the hydrazide resin twice by using DEPC water, adding the oxidized RNA in the step (1), and incubating for 1-2h at 25 ℃;
(3) preparation of N-glycosylated GlycoRNA
Removing the liquid in the centrifugal filter tube in the step (2), washing with 500 mu L DEPC water for three times to ensure that the concentration of RNA in the eluent is 0, and sealing the bottom of the centrifugal filter tube by using a rubber capSeal and add 200. Mu.L 25mM NH to the tube 4 HCO 3 Incubating for 2h at 25 ℃ at ph=7, 10-20 μm PNGase F, collecting eluate and preserving at-80 ℃;
(4) preparation of O-glycosylated GlycoRNA
The filter tube was washed three times with 500. Mu.L DEPC water in step (2) and the concentration of RNA in the eluate was kept at 0, 200. Mu.L 25mM Tris-HCl, pH=7, 10-20 μm GalNAcEXO was added thereto, incubated at 25℃for 1-2 hours, and the eluate was collected and stored at-80 ℃.
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